Prevalence and genotype distribution of HPV6/11/16/18 infections among 180,276 outpatient females from a Women’s and Children’s Central Hospital, 2015–2021, Chengdu, China

The aims of this study on human papilloma virus (HPV) 6/11/16/18 infection among females in Chengdu were to provide more targeted strategies for the prevention and treatment of cervical cancer and genital warts. In this study, the infection status of 20 genotypes was analysed by gene chip technology. The prevalence rates of HPV-6, -11, -16, and -18 infection among 180,276 cases were 0.94%, 0.57%, 3.22%, and 1.28%, respectively. The prevalence of HPV 6/11/16/18 showed a bimodal U-shaped curve with age; the first and second peak occurred among females < 20 and ≥ 60 years old, respectively. As the multiplicity of infections involving HPV6/11/16/18 increases, the infection rate decreases. The ratios of HPV16 single infection showed a yearly increase. The top five genotypes with HPV-16, -18, -6, and -11 in coinfection were HPV52/58/53/51/33, HPV 52/16/53/58/51, HPV52/16/58/51/53 and HPV16/52/58/59/18, respectively, HPV16/18/6/11 were mainly coinfected with HR-HPV. In sum, among the five vaccines available, nonavalent vaccine is more suitable for Chengdu females. For young females prioritizing vaccination is essential in the current context, while HPV screening remains an effective approach for older females. Additionally, in patients with genital warts, it is necessary to assess the presence of high-risk HPV infection and manage it appropriately in patients with genital warts.

screening by doctors.Specific reasons included health examinations, abnormal vaginal bleeding, lower genital tract inflammation, genital warts, infertility, unknown lower abdominal pain, gynaecological tumours, and urethritis.Inclusion criteria consisted of a history of sexual activity, absence from menstruation and pregnancy, while exclusion criteria included women with no sex, menstruating and pregnant women, and women who had undergone uterine surgery.For cases that were reviewed, we only considered the results of the first screening.A total of 6254 cases were excluded from the study.Finally, a total of 180,276 participants were included in this retrospective study conducted at CWCCH between January 2015 and December 2021.The mean age was 38.8 ± 9.1 years (ranging from 12 to 91 years).categorized into age groups of < 20, 20-29, 30-39, 40-49, 50-59,  and ≥ 60.This study was approved by the Medical Ethics Committee of Chengdu Women's and Children's Central Hospital, and all methods were performed in accordance with the relevant guidelines and regulations.

Ethics statement
This research received approval and a waiver for participant informed consent from the Medical Ethics Committee of Chengdu Women's and Children's Central Hospital due to the retrospective nature of the study, where patient identities were deliberately anonymized, rendering individual informed consent unnecessary.

Specimen collection
A gynaecologist wipes cervical secretions with a cotton swab before sampling, places the cervical brush head on the cervix and rotates the brush head clockwise 5 times to obtain a sufficient amount of cervical epithelial cells.Then, the cervical brush head is placed into the sample tube marked with the patient's name, the cap is tightened, and the sample is quickly sent for examination.

DNA extraction and HPV genotyping
DNA extraction and HPV genotyping were performed using an HPV genotyping test kit (Shenzhen GL Bio-Tech Co., Ltd.).The kit detected 20 types of HPV: HPV6, 11, 42, 43, 16, 18, 31,33, 35, 39, 45, 51, 52, 53.56, 58,  59, 66, 68, and 73.The process involved three steps: HPV-DNA extraction, PCR amplification and HPV-DNA hybridization.The Haema9600 (Zhuhai XZ Bio-Tech Co., Ltd.) was used for gene amplification.The amplification parameters were set as follows: ①50 °C for 2 min; ②95 °C for 10 min; ③40 cycles were performed at 95 °C for 30 s, 52 °C for 45 s and 65 °C for 30 s; and ④ 65 °C for 5 min.GL-HB-9600 (Shenzhen GL Bio-Tech Co., Ltd.) was used for DNA hybridization.After color rendering, positive detection results were indicated by clear blue dots.To ensure the reliability of each HPV test result, six probe arrays (including 3 positive anchor points, 1 negative anchor point, and 2 internal control points) were used as the internal control (IC), and HPV16 was added as a positive control.The testing doctor strictly follows the HPV interpretation rules to issue the patient's HPV test results.This study completely ruled out the impact of reproductive pathogens (BV, TV, VVC, NG, CT, etc.) other than HPV on the HPV detection results.Specific HPV extraction solution was added during the DNA extraction stage to ensure specificity.During the DNA amplification stage, HPV primers and probes were added to the reaction mixture to ensure the specific amplification of HPV fragments.The probes on the gene chip were specifically designed for HPV genotypes, ensuring no cross-reactivity between different genotypes during the DNA hybridization process.

Statistical analysis
Microsoft's Excel 2021 was used to process and analyse the data, the chi-square test was used to compare the sample rate between the groups, SPSS 26 (SPSS Inc., Chicago, IL, USA) was used to calculate chi-square data and P values, and two-sided P values of less than 0.05 were considered statistically significant.

Age-specific prevalence of HPV6/11/16/18 infection
The 30-39 years group made up the largest proportion of screened participants, accounting for 38.54% of the total population, yet the < 20 and ≥ 60 age groups made up fewer proportions, accounting for 0.44% and 14.4%, respectively (Table 1).

Discussion
To date, vaccination and HPV screening for women constitute two major strategies in the prevention of cervical cancer and genital warts.The focus of HPV vaccine research lies within the L region 31 and the currently available HPV vaccines are designed using a combination of multiple subtypes of L1 virus-like particles (L1-VLPs), and the L1 spontaneously formed VLPs are highly immunogenic and produce high titers of neutralizing antibodies to prevent HPV infection 14,32 .VLPs mimic the organization and conformation of authentic native viruses but lack the viral genome, inducing an immune response without causing disease 33 .In contrast, attenuated or inactivated viruses may pose risks of incomplete attenuation or reversion to virulence, raising safety concerns.As a result, current HPV vaccines are safer than attenuated or inactivated viruses.A decade of HPV vaccination practice has demonstrated that HPV vaccine can reduce HPV infection rates, genital warts, low-grade and high-grade cervical intraepithelial neoplasia (CIN), and CIN2+ 27 , moreover it has a strong group effect or indirect protection against unvaccinated females 26 .In terms of HPV screening, HPV DNA has a high sensitivity and negative predictive value (fewer high-grade lesions in the subsequent third year of screening) and is an effective screening method 34 .In this study, the prevalence rates of HPV-16 and -18 infections in Chengdu were 3.22% and 1.28%, respectively.These rates were significantly higher than those of the less developed regions of Guangxi 35 (2.7% and 1.11%, respectively) and the more developed regions of Shanghai 36 (2.34% and 1.0%, respectively) but lower than those of the economically comparable Tianjin 37 (5.36% and 1.57%, respectively).The prevalence rates of HPV-6 and -11 infections in Chengdu (0.94% and 0.57%, respectively) were lower than those in Shanghai 36 (1.29% and 0.81%, respectively) and Guangxi 35 (1.31% and 0.82%, respectively) but higher than those in Tianjin 37 (0.30% and 0.51%, respectively).Disparities in HPV infection rates across different regions may be influenced by factors such as local economic conditions, awareness of prevention, lifestyle habits, and HPV detection methods.However, regardless, given the crucial roles of HPV 16/18 and HPV 6/11 in cervical cancer and genital warts, the aforementioned findings underscore the imperative need to enhance HPV vaccine coverage in the Chengdu region.In terms of infection patterns, the ratios of HPV-6, -11, -16 and -18 single infection were 48.56%, 57.41%, 60.71% and 56.23%, respectively, which indicates that single infection is the primary mode of HPV infection, followed by double infection.As the multiplicity of coinfection increased, the prevalence of multiple infections gradually decreased.The occurrence of multiple infections is associated with having multiple sexual partners.The potential impact of multiple infections on cervical cancer risk remains debatable 38,39 .In this study, the top five genotypes in 180,276 cases were HPV-52,-16,-58,-53,-51, and the top five genotypes with HPV16 and HPV18 coinfection were HPV52, -58, -53, -51, -33 and HPV52, -16, -53, -58, -51, respectively, while the top five genotypes with HPV6 and HPV11 coinfection were HPV-52, -16, -58, -51, -53 and HPV-16, -52, -58, -59, -18, respectively.Thus it can be seen that HPV-52 and -58 are the top two genotypes among females in Chengdu.However, currently, among the five available HPV vaccines in the China mainland, only the nonavalent vaccine covers these two genotypes, so the nonavalent vaccine is more suitable for Chengdu females.These findings of this large-scale epidemiological investigation based on HPV genotyping screening hold significance for the prevention and control of diseases such as cervical cancer and genital warts in Chengdu.Additionally, they provide valuable insights for the future design of HPV vaccines.
HPV16, as a highly carcinogenic genotype, was found to have the highest infection rates in HPV6/11/16/18.The single infection positivity rate of HPV16 showed a continuous increase yearly, and studies 40 have indicated that the proportion of high-grade cervical lesions among HPV16 single infections is the highest.In response to the increasing trend of HPV16 single infections, a stronger push for vaccines covering HPV 16 is warranted.www.nature.com/scientificreports/While the infection rate of HPV18 is lower than that of HPV16, its coinfections with HPV52, -58, -53, -51, and -33 genotypes in multiple infections probably enhance its carcinogenic potential.
In this study, the coinfection of HPV6 and HPV11 was very low (42 cases), the single infection of HPV6 and HPV11 accounted for the majority (48.56% and 57.41%, respectively), and most of them had multiple coinfections with HR-HPV.In cases of multiple infections involving HPV 6, the most frequently cooccurring genotype is HPV-52, -16, -58, -51, and -53, whereas for HPV 11, the most commonly cooccurring genotype is HPV-16, -52, 58, -59, and -18.It can be seen that low-risk HPV6 and HPV11 are the most common co-infected LR-HPV with HR-HPV.Studies 41 have pointed out that the most common HPV genotypes in genital warts in China are HPV-6, HPV-11 and HPV-16.In this study, HPV16 ranked second in HPV6 multiple infections (172 coinfections), and HPV16 ranked first in HPV11 multiple infections (97 coinfections), which also explains why HPV16 is the most frequent HR-HPV in genital warts.Moreover, multiple infections of HPV6/11 with HR-HPV may be the cause of genital warts appearing in LSIL, HSIL and tumour progression 42 ; therefore, in patients with genital warts, it is necessary to test whether there is an HR-HPV infection and to address it appropriately.
In this study, the prevalence of HPV6/11/16/18 was bimodal with age, with young women (< 20 years old) having the highest prevalence, old women (≥ 60 years old) having higher prevalence, and middle-aged women (20-49 years old) having a modest decline in prevalence.In fact, HPV infection is bimodal with age and is very common in many regions, and almost all young women have the first peak, with the second peak occurring at different ages [35][36][37]43,44 . The frst peak of HPV infection rates in young women can be up to 80% in some populations 45 , with most women suffering from transient infections that clear rapidly 46 .The highest infection rate among young women (< 20 years old) may be related to less mature immune protection and/or multiple sexual partners in this age bracket 47 but also to the fact that HPV screening is performed only if the patient has a sexual history and associated symptoms (e.g., genital warts) or if the patient requests it, which results in a relatively high detection rate.In the face of the highest HPV6/11/16/18 infection rate among young girls, it is urgent to strengthen HPV vaccination among women who have not had sex before in Chengdu.After this first peak, the prevalence of infection gradually declined to a plateau in middle-aged women (20-49 years old), which is probably due to increased autoimmune function and stable sexual partners.For the second minor peak of infection, the higher rate of infection in old women (≥ 50 years old) may be due to immunosenescence, changes in sexual behaviour and sexual partner (both for male and female), and reappearance of past (latent) infections 44,[48][49][50][51] .Some scholars 52 point out that the geographic difference in this second peak may be partially explained by indirect indicators of menopausal hormonal patterns, such as body mass index (BMI) and ethnicity. Sice young women (age ≤ 20 years) are HPV-susceptible because of high HPV infection rates, vaccination of women before sexual debut is the most cost-effective strategy to prevent cervical cancer.Data 53,54 showed that the earlier young women were inoculated with the HPV vaccine, the higher the antibody titre and the better the protective effect of the vaccine.Studies 55 showed that almost everywhere, most men and women started having sex in their late teens (ages 15-19), and most began having sex at 15. Th crude median sexual debut age for the youngest age group was 17 years in China 56 .Considering Chinese cultural traditions and social factors such as nine-year compulsory education, Chinese scholars recommend that junior middle school girls (aged 13 to 15) to be the first group to be vaccinated.In mainland China, there is an ongoing initiative to gradually introduce free HPV vaccination services or provide subsidized HPV vaccine inoculation.Currently, 19 provinces, municipalities, or autonomous regions across China offer free HPV vaccine administration.As of now, locations within Sichuan Province where free HPV vaccination can be arranged for those junior middle school girls (aged 13 to 15) include the entire city of Chengdu, as well as Wenchuan County in Aba and the city of Barkam, the latter two being economically disadvantaged areas.Similarly, HPV vaccination is also necessary in women from less than the age of 20 to 45, and even in women already infected with HPV, data 54 show that the immune response to the HPV vaccine appears to prevent reinfection or reactivation of disease with vaccine HPV type.Moreover, HPV 16 and 18 vaccines were well tolerated and highly efficacious against HPV 16 and 18-associated high-grade genital lesions and persistent infection 57 .
For the second minor peak of HPV infection, HPV and cytological screening is especially important in middle-aged and older women, as screening not only reduces the burden of precancerous lesions and related persistent HPV infections, but removal of lesions may have a direct antigen-presenting effect that could protect  59 specifically emphasized that additional evaluation (e.g., colposcopy with biopsy) is necessary even when cytology results are negative if HPV 16 or 18 testing is positive and that additional laboratory testing of the same sample is not feasible, the patient should proceed directly to colposcopy.In sum, vaccination is of extreme importance in the current situation.The WHO global strategy to accelerate the elimination of cervical cancer as a public health problem highlights 90% vaccination coverage for girls younger than 15 years old by 2030 as one of the three targets 60 .For middle-aged and older women, regular HPV screening is a better strategy to prevent cervical cancer and precancerous lesions.
The study possesses several limitations.First, it lacked cell or pathological data from positive cases, rendering comparative statistics unfeasible.Second, female vaccination rates were not taken into account, making it impossible to calculate the specific impact of the vaccine on HPV infection rates.Third, the study's subjects comprised solely patients from hospitals within a 6-year timeframe, which does not represent the typical population of Chengdu.Therefore, these findings are exploratory in nature and merit further investigation.Fourth, only four genotypes were involved in this study, obviously, some other HR-HPVs and LR-HPVs are involved in malignant disorders, especially cervical cancer and other genital cancers, However, due to the need for detailed and in-depth study of the four genes, few other genotypes are taken in account, which need to be further presented in future studies.

Conclusions
In conclusion, the infection rate of HPV6/11/16/18 was high in Chengdu, with HPV16 exhibiting the highest infection rate and HPV16 single infections showing an annual increase.The prevalence of HPV6/11/16/18 infection in young women (< 20 years) was highest and in old women (≥ 60 years) was higher; therefore, prioritizing vaccination for young females is essential in the current context, while HPV screening remains an effective approach for older females.Among the available HPV vaccines in Chengdu, the nonavalent vaccine appears to be relatively most suitable for Chengdu females.Additionally, in patients with genital warts, it is necessary to assess the presence of HR-HPV infection and manage it appropriately.

Figure 1 .
Figure 1.Prevalence and genotype distribution of HPV infection in 180,276 women from 2015 to 2021 (HR-HPV and LR-HPV).

Table 2 .
Prevalence of HPV6/11/16/18 infection patterns in different age groups and in every year.

specific prevalence of HPV6/11/16/18 infection
From 2015 to 2021, HPV16 consistently exhibited the highest infection rate (Table1).With the exception of the five age distribution curves of HPV-6, -11, and -18 in 2015 and HPV-18 in 2018 and 2021 (the second peak was not pronounced), the age distribution curves of HPV-6, -11, -16, and -18 from 2015 to 2021 displayed a U-shaped .From 2015 to 2020, the annual infection rates of HPV6 significantly differed with age (P < 0.05).The infection rates of HPV11 were significantly different with age in 2015, 2016, 2018, and 2019 (P < 0.05).The age-related infection rates of HPV16 were significantly different in 2015, 2017, and 2021 (P < 0.05), and the age-related infection rates of HPV18 showed significant differences in 2015 and 2016 (P < 0.05) (Table

Table 4 .
Top 10 genotypes in multiple infections with HPV6/11/16/18 in 2015-2021.*This means that the number of adjacent genotypes is the same, and their sequences are the same.